Current studies to elucidate the functional significance of the nanofiber-based tissue engineering construct in a complex in vivo bone healing microenvironment are limited due to the lack of an appropriate in vivo bone healing model that permits dynamic and high-resolution analysis. To overcome this limitation, we propose to develop a multiphoton laser-scanning microscopy (MPLSM)- based intravital imaging modality that permits dynamic and longitudinal imaging analyses of nanofiber-based tissue engineering constructs. We will develop a calvarial defect window chamber model in mice, which allows intravital microscopy to monitor the cellular constructs in time-lapse imaging series and in real-time functional analysis. Within the proposed two-year funding period, we will establish a reproducible in vivo assay system with defined imaging analysis protocols for quantitative and qualitative evaluation of the bone regenerative properties of the nanofibrous scaffold. The proposal will center on three important design considerations to address nanofiber-mediated repair: ECM organization, cell migration and vascular ingrowth. Our current proposal represents the first attempt to establish such an in vivo assay system to address the role of nanofibers in a complex in vivo healing microenvironment. The success of the proposed study not only could provide a useful tool for the optimization of nanofibrous-based tissue engineering construct, but also potentially offer a biomimetic flexible construct to treat patients with impaired healing. The success of this system could open up experimental evaluation of specific mechanisms for cell-based bone regeneration, further offering an experimental testing modality for therapeutic strategies. PUBLIC HEALTH RELEVANCE: Nanofiber holds great promise in tissue repair and regeneration due to its versatility in creating an integrated platform for stem cell manipulations. The project will use state-of- the-art imaging technology to define the important elements in the design of nanofibers for bone repair and regeneration.